1. Field of the Invention
The present invention relates to a pharmaceutical preparation, comprising at least one Toll-like receptor ligand and at least one peptide. The invention also relates to the use of such a pharmaceutical preparation and to a vaccination method.
2. Discussion of Background Information
The adaptive immune system plays an important role in immune defence against specific microorganisms, for example viruses, specific fungi and bacteria, as well as in recognising and repelling malignant (degenerate) cells (tumour cells). Here diseased, i.e. virus-infected or malignant, cells are specifically recognised and eliminated by what are known as cytotoxic T cells (CTL, killer cells). A necessary prerequisite for recognition of the diseased cells by the cytotoxic T cells is the interaction of the T cell receptor (TCR) of the cytotoxic cells with a main histocompatibility complex I (MHC I). Protein fragments (from peptides consisting of 8 to 10 amino acids, what are known as MHC class I ligands) are presented on the cell surface by MHC I molecules. These are then specifically recognised by a T cell receptor (TCR). MHC class I ligands are generated in every cell by enzymatic breakdown and are presented on the cell surface (what are known as self-peptides). Virus-specific or tumour-specific self-peptides are also presented on virus-infected or malignant cells. Such cells can thus be recognised and destroyed by cytotoxic T cells (CTL) with a virus-specific or tumour-specific T cell receptor.
To prevent damage to healthy cells, which also present self-peptides, by cytotoxic T cells (CTL), in addition to the specific interaction between T cell receptor and MHC I self-peptide, CTLs must first of all be activated by additional co-stimulatory signals, so they can subsequently recognise and destroy their target cells (virus-infected cells or tumour cells). Without prior activation a cytotoxic cell (CTL), despite its T cell receptor specific to a certain (for example virus-infected or malignant) target cell, cannot recognise and destroy the target cells via the specific self-peptides thereof. If, on the other hand, cytotoxic T cells, which comprise specific receptors for self-peptides presented on cells, are wrongly activated, for example within the framework of an infection, this can lead to reactions of cytotoxic cells against healthy tissue cells. Regulation of the activation of cytotoxic T cells is therefore of prime importance for whether a specific immune response is initiated or not.
On activation of cytotoxic T cells with initiation of a specific immune response what are known as professional antigen-presenting cells (APC) play a particular role. Antigen-presenting cells are specialised cells which absorb and present antigens very efficiently (for example viruses or bacteria). Examples are dendritic cells (DC) or the Langerhans cells in the skin. DCs mature in the presence of danger signals. This leads to a high regulation of co-stimulatory signals. In this situation the recognition of antigens leads to activation of cytotoxic T cells (CTL) and to initiation of the immune response.
For an efficient T cell response to occur the activating interaction between the cytotoxic T cells and professional antigen-presenting cells is therefore necessary, and the specific activation of the T cells takes place owing to two different signals being received, namely the specific recognition of the antigen owing to suitable receptors, and co-stimulatory signals which are imparted by the receptors and messengers of the antigen-presenting cells (APC). The APCs only pass on these co-stimulatory signals in the activated state, however. The activation of the APC can be attained by stimulation of what are known as Toll-like receptors (TLR).
“Danger signals” which lead to maturing of dendritic cells can have different natures. They can for example originate from activated T helper cells or even directly from pathogens. If the “danger signals” originate from pathogens, certain conserved virus, bacteria or fungi structures can, for example, be recognised by the Toll-like receptors.
A total of 11 different Toll-like receptors (TLR) have been known up to now and natural and synthetic ligands are described for many of these [“S. Akira, K. Takeda; Toll-like receptor signalling’ Nat. Rev. Immunol. 2004; 4, 499 to 511”]. For example cell wall components of gram-negative bacteria (lipopolysaccharides; LPS) of TLR4 are recognised. The genomic dsRNSs of certain viruses are recognised by TLR3. Documents WO-A 00/040,228, WO-A 01/015,698 and WO-A 045,494 also describe the recognition of different viral structures, which occur in connection with human and animal diseases, by TLR7, i.e. the substance imiquimod {1-(2-methylpropyl-)1H-imidazo[4,5-c]quinolin-4-amine}.
Despite significant advances in the understanding of the above-described underlying functionality of the adaptive immune response and improved knowledge of the antigen structures that exist on diseased (for example virus-infected) cells or tumour cells and are recognised by specific cell receptors, until now it has only be insufficiently possible to activate the adaptive immune system by therapeutic measures with the aim of specifically eliminating diseased cells or tumour cells.
Vaccinations are a long-known method of activating the immune system. Previously it has been possible to use vaccinations substantially only prophylactically since the administration of a vaccine leads to production of protective antibodies (B cell response). Immunisation takes a relatively long time, usually several weeks, so the therapeutic use of vaccinations (i.e. the rapid activation of the immune system for treating acute states) is largely rejected. The development of strategies in which intervention in the cascade of activation of T cells is possible, thereby generating a T cell response, is therefore of immense interest. In the individual case this could be beneficial therapeutically as well as prophylactically.
Tumour immunology incorporates a wide field of biological phenomena which include the interaction of malignant, degenerate cells with the congenital and adaptive immune system. Among the various types of immune cells T cells play a central role since they can recognise and eliminate specific tumour cells. Since most tumour-associated proteins occur intracellularly, T cells are obviously the most suitable tool for inducing a tumour-specific immune response. In particular antibodies cannot penetrate into the cell and congenital immune cells lack the high-grade specificity of T cells. The above-described activation of cytotoxic T cells, which eliminate only diseased cells, but spare healthy ones, is therefore an attractive aim for the prevention and treatment of infections, specifically virus-induced infections and tumoral diseases.
For the majority of conventional vaccination strategies it is necessary to carry out a plurality of immunisation steps to attain adequate immunity. Immunity usually results from the production of protective antibodies against the antigen administered in the course of vaccination.
Previous strategies for inducing T cells by immunisation used preparations which contain the corresponding antigen in conjunction with auxiliaries/adjuvants, such as aluminium-containing substances or virus-like particles (VLP), incomplete Freund's adjutants or similar oil-in-water mixtures or CpG-DNS. The preparations are applied subcutaneously or intradermally by means of injection. This is disclosed for example in the documents “E. Tartour, F. Benchetrit, N. Haicheur, O. Adotevi and W. H. Fridman; Synthetic and natural non-live vectors: rationale for their clinical development in cancer vaccine protocols; Vaccine, 19 Dec. 2002: 20 (Suppl. 4) A 32 to 39”; “R. Edelmann: Adjuvants for the future; in M. M. Levine, G. C. Woodrow, J. B. Kaper, G. S. Cobon (ed.), New Generation Vaccines, vol. 2, New York, Marcel Dekker (1997), 173 to 192)”; and “S. Agrwal, E. R. Kadnimalia; Modulation of Toll-like Receptor 9 responses through synthetic immunostimulatory motifs of DNA; Ann. N.Y. Acad. Sci. 2003 (December): 1002, 30 to 42”. An attempt has also been made to initiate T cell responses with the aid of dendritic cells, of which the precursor cells have been previously obtained from blood and differentiated in vitro [“T. G. Berger, E. S. Schultz; Dendritic cell-based immunotherapy; Curr. Top. Microbiol. Immunol. 2003: 276, 163 to 197”] or to use dendritic cells as adjutants in that these were charged with MHC class I ligands, tumour-associated proteins or tumour cell lysates and patients were injected [“G. Schuler, B. Schuler-Thurner, R. M. Steinmann; The use of dendritic cells in cancer immunotherapy; Curr. Opin. Immunol. 2003: 15, 138 to 147”].
Owing to the significance of T cells to the elimination of intracellular pathogens and tumour cells, more recent immunisation strategies attempt to activate T cells, in particular cytotoxic T cells, and this would also open up the possibilities of therapeutic vaccinations. Common to all previous vaccination strategies is that they function passably in practice, in other words, that cytotoxic T cells for example can be measurably activated in patients and their number may be increased. However, the methods are only moderately capable of generating efficient T cell responses, in particular of attaining adequate repulsion of tumours. In addition the known vaccination methods are still associated with significant side effects through to toxicity [“E. Tartour et al, loc. cit.”, and R. Edelmann, loc. cit.”]. It is known from animal models that in the case of vaccinations with CpG-DNS side effects occur which considerably limit use of this adjuvant. In addition the DC-based methods in particular are associated with a level of expenditure which cannot be transferred to the daily routine of a hospital or practice.
The object of the invention was to contribute to significantly improved generation of cytotoxic T cells by way of efficient activation without the drawbacks of the prior art occurring in the process. In particular the aim is to achieve efficient activation of antigen-presenting cells via activation of Toll-like receptors. These cells make sure of efficient activation of specific cytotoxic T cells by increased expression of co-stimulatory signals.
A further object of the invention was to disclose a method of non-invasive triggering of a systemic, T cell-imparted immune response.
Yet another object of the invention was to provide an inexpensive vaccination concept, associated with only low technical complexity, for generating cytotoxic T cells.
Yet another object of the invention was to provide preparations for new preventative and therapeutic methods on an immunological basis against a broad spectrum of diseases, including diseases triggered by viruses or bacteria and tumoral diseases.
Surprisingly it has been found according to the invention that topically administered preparations which contain the necessary specific peptide signal substances for the T cell receptor together with a ligand for a Toll-like receptor are non-invasively capable of triggering a systemic T cell imparted immune response in that they are capable of generating specific cytotoxic T cells. A simple, fast and efficient method is thereby disclosed for inducing immune responses to any desired T cell antigen.
The invention therefore relates to a pharmaceutical preparation which comprises at least one Toll-like receptor ligand, at least one peptide and optionally one or more substance(s) from the group comprising carriers, adjuvants and additives.
The invention also relates to the use of a pharmaceutical preparation as described in detail hereinafter for the prophylaxis and/or therapy of diseases in a mammal induced by viruses and/or bacteria and/or fungi and for the preparation of a pharmaceutical composition for the prophylaxis and/or therapy of diseases in a mammal induced by viruses and/or bacteria and/or fungi.
The invention also relates to the use of a pharmaceutical preparation as described in detail hereinafter for the prophylaxis and/or therapy of diseases in a mammal caused by excessive cell proliferation and for the preparation of a pharmaceutical composition for the prophylaxis and/or therapy of diseases in a mammal caused by excessive cell proliferation.
The invention also relates to methods for the prophylaxis and/or therapy of diseases in a mammal induced by viruses and/or bacteria and/or fungi and for the prophylaxis and/or therapy of diseases caused by excessive cell proliferation, comprising the administration of a pharmaceutical preparation, according to the detailed description hereinafter, to the mammal in a concentration of the pharmacologically effective components that is effective for the prophylaxis and/or therapy of said diseases.
The invention also relates to a method for the prophylactic and/or therapeutic activation of the adaptive immune system of a mammal, comprising the administration of a pharmaceutical preparation, as described in detail hereinafter, to the mammal in a concentration of the pharmacologically effective components that is effective for the prophylactic and/or therapeutic activation of the mammal's immune system, and to a method for the prophylactic and/or therapeutic activation of cytotoxic T cells in the immune system of a mammal, comprising the administration of a pharmaceutical preparation, as described in detail hereinafter, to the mammal in a concentration of the pharmacologically effective components that is effective for the prophylactic and/or therapeutic activation of the cytotoxic T cells.